Plant transformation without selection

ABSTRACT

The invention provides methods for identifying regenerated transformed plants and differentiated transformed plant parts, obtained without subjecting plant cells to selective conditions prior to regenerating the cells to obtain differentiated tissues. In particular embodiments, the plant cells are corn plant cells. Methods for growing and handling plants, including identifying plants that demonstrate specific traits of interest are also provided.

This application claims the priority of U.S. provisional applicationSer. No. 60/841,519 filed Aug. 31, 2006, the entire disclosure of whichis incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the field of plant biotechnology. Inparticular, the invention relates to methods for producing transgenicplants not requiring use of a selectable marker gene prior to obtaininga regenerated plant or plant part.

2. Description of the Related Art

Stable transformation of plant cells and production of transgenic plantshas typically required a selection step, wherein plant tissue isselected in the presence of a selection agent after having beencontacted by one or more exogenous nucleic acid sequences, includingones that comprise a sequence or sequences encoding a gene of interestand a marker gene. Following such selection, stably transformed plantscomprising a gene of interest (GOI) may be regenerated and identified.However, upon creating a transformed plant comprising a GOI, aselectable or screenable marker gene which is not itself a GOI istypically no longer necessary, and its presence may complicatesubsequent analyses and product development efforts. Furthermore, thenecessity of a strong promoter to drive a selectable marker has beenshown to bias the expression of the desired gene (Yoo et al., 2005).

A wide range of methods has been reported for creating marker-gene freetransgenic plants, for example co-transformation, transposable elements,site-specific recombination, and intrachromosomal recombination (e.g.Darbani et al., 2007). However most of these systems are time-consumingand inefficient. Goldsbrough (2001) reviews methods for avoiding the useof, or eliminating, selectable marker genes in creating transgenicplants.

De Vetten et al., (2003; and U.S. Patent Application Publication2005/0097641) describe methods for marker-free transformation of avegetatively propagated crop, such as potato, however resulting inchimeric plants. Palys et al. (PCT Publication WO 2004/081184) describetransformation of tomato, lettuce, and cabbage without selection.Francis and Spiker (2005) describe identification of transgenicArabidopsis lines using a PCR-based screen, to avoid selection bias intransgene integration. In contrast, the present invention providesmethods for rapid and efficient production of germline-transformed cornplants obtained via methods not requiring the presence of a selectiveagent or a screenable marker gene, such as a visual marker gene, priorto obtaining regenerated corn plants.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for identifyingtransgenic corn plants, comprising: (a) obtaining corn plant cellstransformed with a DNA segment comprising a nucleic acid sequence ofinterest; (b) regenerating a plurality of corn plants or differentiatedcorn plant parts from the cells without first selecting for the presenceof said DNA segment; and (c) identifying at least a first transgeniccorn plant or differentiated plant part from the plurality of cornplants or differentiated corn plant parts. In some embodiments, the DNAsegment does not comprise a selectable marker gene, or a visual markergene. In other embodiments, the plants are regenerated by growth onsolid media, liquid media, or a combination of solid and liquid media.In particular embodiments, the plants are regenerated by growth solelyon liquid media subsequent to contacting the cells with a GOI and priorto identifying the transgenic corn plant or transgenic differentiatedplant part. In certain embodiments, the transformation frequency ofcells grown solely in liquid media subsequent to contacting the cellswith a GOI and prior to identification of transgenic plants ortransgenic plant parts is enhanced relative to the transformationfrequency observed when cells are grown in solid media or soilsubsequent to contacting the cells with a GOI and prior toidentification of transgenic plants or transgenic plant parts.

In certain embodiments, the plant cells are immature corn embryo cells.In particular embodiments the immature corn embryos are from about 1.5mm to about 3.5 mm in length, or from about 1.9 mm to about 2.3 mm inlength.

In certain embodiments, the method further comprises, between steps (b)and (c), (1) placing the plurality of corn plants or differentiatedplant parts in culture tubes or growth plugs comprising a growth mediumor water while maintaining the individual identity of the corn plants;and (2) subjecting the plants or plant parts to at least a first assayfor the presence of the DNA segment to identify one or more plant orplant part as transgenic based on results from the assay. The assay mayfurther be selected from the group consisting of Southern hybridization,PCR, DNA sequencing, northern blotting, western blotting, animmunoassay, and an assay for the enzymatic activity encoded by the DNAsegment. In particular embodiments the assay is performed prior toplacing the regenerated plants into soil. In other embodiments, theputatively transformed corn plants or differentiated plant parts lackingthe nucleic acid sequence of interest are identified, wherein the assayis performed on plant tissue comprising pooled subsets of nucleic acidsfrom said plurality of corn plants or differentiated plant parts.

In some embodiments, the corn plants or corn plant parts are regeneratednot later than 6 weeks after the DNA segment is transformed into thecorn plant cells. In other embodiments, the corn plants or corn plantparts are regenerated not later than 4 weeks after the DNA segment istransformed into the corn plant cells. In yet other embodiments, thecorn plants or corn plant parts are regenerated not later than 3 weeksafter the DNA segment is transformed into the corn plant cells. In stillyet other embodiments, the corn plants or corn plant parts areregenerated not later than 2 weeks after the DNA segment is transformedinto the corn plant cells. In further embodiments, the corn plants orcorn plant parts are regenerated not later than 1 week after the DNAsegment is transformed into the corn plant cells.

In certain embodiments, the DNA segment is introduced into the cornplant cell by bacterially-mediated transformation, electroporation,PEG-mediated transformation, or particle bombardment. In particularembodiments, the bacterially-mediated transformation is mediated by abacterial cell selected from the group consisting of an Agrobacteriumcell, a Rhizobium cell, a Sinorhizobium cell, and a Mesorhizobium cell.

The method may further comprise the step of subjecting a corn plant orplant part derived from the first corn plant cell to culture conditionsthat select for, or allow screening for, the presence or absence of thenucleic acid sequence of interest after regeneration of a plant or plantpart. In certain embodiments, the growth medium is a solid medium. Inyet other embodiments, the growth medium is liquid. In still otherembodiments, the growth medium is soil. In other embodiments theregenerated plant or differentiated plant part is uniform with respectto the presence of the DNA segment.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 Schematic comparison of no selection (“no sel”) and 2Ttransformation protocols. (For further details on 2T strategy, pleasesee Huang et al 2004)

FIG. 2 Histochemical analysis using GUS of representative regeneratinglines.

FIG. 3 Recovery of glyphosate-tolerant events through “no selection”approach after spraying the plants with glyphosate solution.

FIG. 4 Representative Southern analysis data from selected R₀ plants asdescribed in Example 4.

FIG. 5 Germline transmission and segregation of GUS gene to nextgeneration was validated using R₀ pollen and backcrossing with parentalline.

FIG. 6 Southern analysis data from progeny of selected independenttransformation events, demonstrating stable transmission of transformedsequences (CP4 gene).

FIG. 7 Arrangement of growth plugs to allow easy identification ofindividual plants following assay for presence of transgenic sequence.

FIG. 8 Schematic diagram of regeneration protocols utilizing semi-solidmedia-comparing selection and no selection approaches.

FIG. 9A and FIG. 9B Regeneration protocols using liquid culture withoutselection prior to obtaining a regenerated plant-comparison ofproliferation media.

DETAILED DESCRIPTION OF THE INVENTION

Development of many modern genetically transformed plant productsinvolves stacking of multiple transgenic traits together to providemultiple value-added traits to farmers. A major bottleneck in thisprocess is the presence of selectable marker genes, which are carriedalong with a gene of interest (GOI) during transformation, as theprocess has typically relied on the use of a selectable marker gene toensure transformation of plant cells. Although various methods areavailable for removing selectable marker genes following transformation,these methods are often time consuming and not highly efficient.

The present invention eliminates the aforementioned bottleneck throughthe development of an efficient transformation process without requiringthe use of a selectable marker gene, as well as efficient plant handlingand screening methods for advancing transgenic events produced withoutselection. In particular, the invention relates to methods for improvingplant transformation efficiency and subsequent regeneration withoutusing selection, leading to the production of marker-free transgenicevents. This is a significant breakthrough in the production oftransgenic crop plants, as marker-less transformation (as well assubsequent regeneration of plants in the absence of a selective agent)avoids the complexity associated with marker removal, avoids biasing thegenetic structure of resultant transformation events due to a need forinitial expression of a selectable marker gene, and also avoidspotential difficulties during the progeny advancement process (e.g. dueto segregation of transgenes corresponding to a GOI relative to thosethat encode a selectable marker). The process also eliminates the needfor use of an additional expression cassette for the selectable orscreenable marker gene, thereby reducing the size of the transformationvector and providing associated benefits such as reducing the chances ofsilencing due to repetitive cassette sequences, promoter interferenceand simplified construction of transformation vectors.

High-throughput production of selectable marker-free transgenic plantsrequires efficient production of transformants. It is preferable thattransformation without selection, more specifically without selectionprior to obtaining regenerated shoots or whole plantlets (comprisingshoots and roots), be carried out in the absence of a selective agent.In certain embodiments, the nucleic acid sequences transformed into atarget plant cell may comprise no selectable marker gene. In otherembodiments, a selectable marker gene or visual marker gene may bepresent, but the transformed cells and regenerating tissues arenevertheless not subjected to a selective agent to which the selectablemarker gene specifies tolerance, resistance, or other assayablephenotype.

Further, these transformants are preferably non-chimeric (i.e. uniform)with respect to the presence of a GOI, since the presence of chimericplant tissues, that are non-uniform with respect to the presence of aGOI, complicates further analysis, production, and identification ofprogeny plants comprising the GOI. It has thus been found thattransformation, including subsequent regeneration steps, withoutselection to routinely produce non-chimeric transgenic plants requiresefficient production of large transgenic sectors and rapid production ofshoot primordia.

Further, in the absence of selective pressure, large numbers of plantsmay be regenerated, many or most of which lack a GOI. Thus, efficientmethods for regenerating, growing, and identifying plants potentiallycomprising a GOI are provided. In certain embodiments, regeneration ofplants is performed in a semi-solid medium prior to transplanting ofputative transformants into soil. In other embodiments, the media may beliquid. In yet other embodiments, a combination of semi-solid and liquidmedia may be employed during the regeneration process, to facilitateplant handling, and to save time, money, and expense, during screeningand transfers to the different growth conditions utilized during thetissue culture process. In still yet other embodiments, only liquidmedia are used during the regeneration process. In particularembodiments, regeneration on liquid media may enhance the transformationfrequency of the cells contacted by a gene of interest.

The presence of a selective agent throughout the tissue culture stepsleading to a regenerated transformed plant may bias the characteristicsof the selected tissue, essentially by requiring a certain level ofexpression of the selectable marker in order for tissue to survive theselective pressure. This may result, for instance, in a bias towardobtaining transgenic events with multiple or complex insertions of aheterologous nucleic acid sequence. Thus, the invention provides amethod for obtaining a population or series of putatively transformedplants without the plants having been subjected to such a selectivepressure during phases of tissue culture such as callus proliferation,pre-regeneration, and regeneration, and which plants may display anadvantageous expression profile of a GOI, and/or advantageouscharacteristics relating to the molecular structure and geneticsegregation of the transgene insertion site(s) found in a given event.In particular, such an advantageous characteristic may include, forinstance, that a significant proportion of transformation eventsdisplays an advantageous level of expression of a GOI, or that asignificant proportion of transformation events displays low copy number(i.e. 1-2 copies) insertions. In particular embodiments, the low copynumber transformation events lack oriV or other vector backbonesequences, if such sequences were present in the original transformationconstruct that initially contacted plant cells at the start of thetransformation process.

Transformation and regeneration without such selection, in accordancewith the methods of the present invention, is reproducible andefficient. In certain embodiments, the transformation frequency (TF), asexpressed for instance on the basis of the number of stably transformeduniform (i.e. non-chimeric) plants obtained, per immature embryo orother explant comprising cells contacted by a heterologous nucleic acidconstruct, is at least 3%, and may range from about 3% to about 60%depending upon the embryo size and cultural conditions including type ofregeneration regeneration methods. In particular embodiments, the TF mayrange from about 10% to about 15%. Alternatively, TF may be calculatedin other ways, for instance based on the number of transformed plantsobtained, per number of plants regenerated and grown from such immatureembryos or other explants.

In certain embodiments, the crop plant being transformed withoutselection is selected from among monocot crop plants, including thePoaceae, such as corn, rice, sorghum, wheat, rye, millet, sugarcane,oat, triticale, turfgrass, and switchgrass plants. In a particularembodiment, the crop plant is a corn (maize) plant. In certainembodiments, the transformation target tissue, e.g. explant, contactedby a heterologous nucleic acid sequence comprises meristematic tissue,such as an embryo, or a shoot meristem. In certain embodiments theexplant is an embryo. In particular embodiments, the embryo is animmature embryo. In still further embodiments, the immature embryo is animmature corn embryo, and is between about 1.9 and 3.5 mm in size, orbetween about 1.6-1.8 mm in size. In particular embodiments, theimmature corn embryo is between about 1.9 and 2.5 mm, and preferablyabout 2.3 mm in size. In other embodiments, the immature corn embryo isabout 2.5-3.2 mm in size, or about 2.8-4.0 mm in size. The immatureembryo may also be selected as a transformation target based on itsdevelopmental stage, or the timing of its isolation, days afterpollination (DAP), for instance about 9-14 days DAP, or about 10-12 DAP.

To initiate a transformation process in accordance with the presentinvention, it is first necessary to select genetic components to beinserted into the plant cells or tissues. Genetic components can includeany nucleic acid that is introduced into a plant cell or tissue usingthe method according to the invention. Genetic components can includenon-plant DNA, plant DNA or synthetic DNA.

In a preferred embodiment, the genetic components are incorporated intoa DNA composition such as a recombinant, double-stranded plasmid orvector molecule comprising at least one or more of following types ofgenetic components: (a) a promoter that functions in plant cells tocause the production of an RNA sequence, (b) a structural DNA sequencethat causes the production of an RNA sequence that encodes a product ofagronomic utility, and (c) a 3′ non-translated DNA sequence thatfunctions in plant cells to cause the addition of polyadenylatednucleotides to the 3′ end of the RNA sequence.

The vector may contain a number of genetic components to facilitatetransformation of the plant cell or tissue and regulate expression ofthe desired gene(s). In one preferred embodiment, the genetic componentsare oriented so as to express an mRNA, which in one embodiment can betranslated into a protein. The expression of a plant structural codingsequence (a gene, cDNA, synthetic DNA, or other DNA) that exists indouble-stranded form involves transcription of messenger RNA (mRNA) fromone strand of the DNA by RNA polymerase enzyme and subsequent processingof the mRNA primary transcript inside the nucleus. This processinginvolves a 3′ non-translated region that adds polyadenylated nucleotidesto the 3′ ends of the mRNA.

Methods for preparing plasmids or vectors containing the desired geneticcomponents are well known in the art. Vectors typically consist of anumber of genetic components, including but not limited to regulatoryelements such as promoters, leaders, introns, and terminator sequences.Regulatory elements are also referred to as cis- or trans-regulatoryelements, depending on the proximity of the element to the sequences orgene(s) they control.

Transcription of DNA into mRNA is regulated by a region of DNA usuallyreferred to as the “promoter”. The promoter region contains a sequenceof bases that signals RNA polymerase to associate with the DNA and toinitiate the transcription into mRNA using one of the DNA strands as atemplate to make a corresponding complementary strand of RNA.

A number of promoters that are active in plant cells have been describedin the literature. Such promoters would include but are not limited tothe nopaline synthase (NOS) and octopine synthase (OCS) promoters thatare carried on tumor-inducing plasmids of Agrobacterium tumefaciens, thecaulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19Sand 35S promoters and the figwort mosaic virus (FMV) 35S promoter, theenhanced CaMV35S promoter (e35S), the light-inducible promoter from thesmall subunit of ribulose bisphosphate carboxylase (ssRUBISCO, a veryabundant plant polypeptide). All of these promoters have been used tocreate various types of DNA constructs that have been expressed inplants.

Promoter hybrids can also be constructed to enhance transcriptionalactivity (U.S. Pat. No. 5,106,739), or to combine desiredtranscriptional activity, inducibility and tissue specificity ordevelopmental specificity. Promoters that function in plants include butare not limited to promoters that are inducible, viral, synthetic,constitutive as described, and temporally regulated, spatiallyregulated, and spatio-temporally regulated. Other promoters that aretissue-enhanced, tissue-specific, or developmentally regulated are alsoknown in the art and envisioned to have utility in the practice of thisinvention.

Promoters may be obtained from a variety of sources such as plants andplant DNA viruses and include, but are not limited to, the CaMV35S andFMV35S promoters and promoters isolated from plant genes such asssRUBISCO genes. As described below, it is preferred that the particularpromoter selected should be capable of causing sufficient expression toresult in the production of an effective amount of the gene product ofinterest.

The promoters used in the DNA constructs (for example,chimeric/recombinant plant genes) of the present invention may bemodified, if desired, to affect their control characteristics. Promoterscan be derived by means of ligation with operator regions, random orcontrolled mutagenesis, etc. Furthermore, the promoters may be alteredto contain multiple “enhancer sequences” to assist in elevating geneexpression.

An mRNA produced by a DNA construct of the present invention may alsocontain a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene and can bespecifically modified so as to increase translation of the mRNA. The 5′non-translated regions can also be obtained from viral RNAs, fromsuitable eukaryotic genes, or from a synthetic gene sequence. Such“enhancer” sequences may be desirable to increase or alter thetranslational efficiency of the resultant mRNA. The present invention isnot limited to constructs wherein the non-translated region is derivedfrom both the 5′ non-translated sequence that accompanies the promotersequence. Rather, the non-translated leader sequence can be derived fromunrelated promoters or genes (see, for example U.S. Pat. No. 5,362,865).Other genetic components that serve to enhance expression or affecttranscription or translational of a gene are also envisioned as geneticcomponents.

The 3′ non-translated region of the chimeric constructs should contain atranscriptional terminator, or an element having equivalent function,and a polyadenylation signal that functions in plants to cause theaddition of polyadenylated nucleotides to the 3′ end of the RNA.Examples of suitable 3′ regions are (1) the 3′ transcribed,non-translated regions containing the polyadenylation signal ofAgrobacterium tumor-inducing (Ti) plasmid genes, such as the nopalinesynthase (NOS) gene, and (2) plant genes such as the soybean storageprotein genes and the small subunit of the ribulose-1,5-bisphosphatecarboxylase (ssRUBISCO) gene. An example of a preferred 3′ region isthat from the ssRUBISCO E9 gene from pea (European Patent Application0385 962).

Typically, DNA sequences located a few hundred base pairs downstream ofthe polyadenylation site serve to terminate transcription. The DNAsequences are referred to herein as transcription-termination regions.The regions are required for efficient polyadenylation of transcribedmessenger RNA (mRNA) and are known as 3′ non-translated regions. RNApolymerase transcribes a coding DNA sequence through a site wherepolyadenylation occurs.

In one embodiment, the T-DNA does not comprise a selectable, screenable,or scoreable marker gene. Alternatively, the DNA to be transferred maycontain a selectable, screenable, or scoreable marker gene, although incertain embodiments of the invention plant tissues are only selected orscreened for the presence of the marker after regeneration has occurred.These genetic components are also referred to herein as functionalgenetic components, as they produce a product that serves a function inthe identification of a transformed plant, or a product of agronomicutility. The DNA that serves as a selection device functions in aregenerable plant tissue, in particular a regenerated tissue, to producea compound that would confer upon the plant tissue resistance to anotherwise toxic compound. Genes of interest for use as a selectable,screenable, or scoreable marker would include but are not limited touidA encoding GUS, gfp, encoding green fluorescent protein (GFP),anthocyanin biosynthesis related genes (C1, B-peru), luciferase (LUX),and genes specifying resistance to antibiotics like kanamycin (Dekeyseret al., 1989), and herbicides like glyphosate (Della-Cioppa et al.,1987). Other selection methods can also be implemented including but notlimited to tolerance to phosphinothricin, bialaphos, and positiveselection mechanisms and would still fall within the scope of thepresent invention.

The present invention can be used with any suitable plant transformationplasmid or vector containing a selectable or screenable marker andassociated regulatory elements as described, along with one or morenucleic acids expressed in a manner sufficient to confer a particulartrait. Examples of suitable structural genes of agronomic interestenvisioned by the present invention would include but are not limited togenes for insect or pest tolerance, herbicide tolerance, genes forquality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology or plant product(s).

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see for example, Birdet al., 1991). The RNA could also be a catalytic RNA molecule (forexample, a ribozyme) engineered to cleave a desired endogenous mRNAproduct (see for example, Gibson and Shillitoe, 1997). Moreparticularly, for a description of anti-sense regulation of geneexpression in plant cells see U.S. Pat. No. 5,107,065 and for adescription of gene suppression in plants by transcription of a dsRNAsee U.S. Pat. No. 6,506,559, U.S. Patent Application Publication No.2002/0168707 A1, and U.S. patent application Ser. Nos. 09/423,143 (seeWO 98/53083), 09/127,735 (see WO 99/53050) and 09/084,942 (see WO99/61631), all of which are incorporated herein by reference. Thus, anygene that produces a protein or mRNA that expresses a phenotype ormorphology change of interest is useful for the practice of the presentinvention.

Exemplary nucleic acids that may be introduced by the methodsencompassed by the present invention include, for example, DNA sequencesor genes from another species, or even genes or sequences that originatewith or are present in the same species, but are incorporated intorecipient cells by genetic engineering methods rather than classicalreproduction or breeding techniques. However, the term exogenous is alsointended to refer to genes that are not normally present in the cellbeing transformed, or perhaps simply not present in the form, structure,etc., as found in the transforming DNA segment or gene, or genes thatare normally present yet that one desires, for example, to haveover-expressed. Thus, the term “exogenous” gene or DNA is intended torefer to any gene or DNA segment that is introduced into a recipientcell, regardless of whether a similar gene may already be present insuch a cell. The type of DNA included in the exogenous DNA can includeDNA that is already present in the plant cell, DNA from another plant,DNA from a different organism, or a DNA generated externally, such as aDNA sequence containing an antisense message of a gene, or a DNAsequence encoding a synthetic or modified version of a gene.

Technologies for the introduction of DNA into cells are well known tothose of skill in the art and can be divided into categories includingbut not limited to: (1) chemical methods; (2) physical methods such asmicroinjection, electroporation, and micro-projectile bombardment; (3)viral vectors; (4) receptor-mediated mechanisms; and (5)Rhizobia-mediated (e.g. Agrobacterium-mediated) plant transformationmethods (e.g. Broothaerts et al., 2005).

For Agrobacterium-mediated transformation, after the construction of theplant transformation vector or construct, said nucleic acid molecule,prepared as a DNA composition in vitro, is introduced into a suitablehost such as E. coli and mated into another suitable host such asAgrobacterium, or directly transformed into competent Agrobacterium.These techniques are well-known to those of skill in the art and havebeen described for a number of plant systems including soybean, cotton,and wheat (see, for example U.S. Pat. Nos. 5,569,834 and 5,159,135, andWO 97/48814, herein incorporated by reference in their entirety).

The present invention encompasses the use of bacterial strains tointroduce one or more genetic components into plants. Those of skill inthe art would recognize the utility of Agrobacterium-mediatedtransformation methods in such a process. A number of wild-type anddisarmed strains of Agrobacterium tumefaciens and Agrobacteriumrhizogenes harboring Ti or Ri plasmids can be used for gene transferinto plants. Preferably, the Agrobacterium hosts contain disarmed Ti andRi plasmids that do not contain the oncogenes that cause tumorigenesisor rhizogenesis, respectively, which are used as the vectors and containthe genes of interest that are subsequently introduced into plants.Preferred strains would include but are not limited to Agrobacteriumtumefaciens derived from strain C58, a nopaline-type strain that is usedto mediate the transfer of DNA into a plant cell, octopine-type strainssuch as LBA4404 or succinamopine-type strains, for example, EHA101 orEHA105. Other bacteria such as Sinorhizobium, Rhizobium, andMesorhizobium that interact with plants naturally can be modified tomediate gene transfer to a number of diverse plants. Theseplant-associated symbiotic bacteria can be made competent for genetransfer by acquisition of both a disarmed Ti plasmid and a suitablebinary vector (Broothaerts et al, 2005). The use of these strains forplant transformation has been reported and the methods are familiar tothose of skill in the art.

The explants can be from a single genotype or from a combination ofgenotypes. Any corn seed that can germinate is a viable startingmaterial. In a preferred embodiment, superior explants from planthybrids can be used as explants. For example, a fast-growing cell linewith a high culture response (higher frequency of embryogenic callusformation, growth rate, plant regeneration frequency, etc.) can begenerated using hybrid embryos containing several genotypes. In oneembodiment, an F₁ hybrid or first generation offspring of cross-breedingcan be used as a donor plant and crossed with another genotype. Those ofskill in the art are aware that heterosis, also referred to herein as“hybrid vigor”, occurs when two inbreds are crossed. The presentinvention thus encompasses the use of an explant resulting from athree-way cross, wherein at least one or more of the inbreds is highlyregenerable and transformable, and the transformation and regenerationfrequency of the three-way cross explant exceeds the frequencies of theinbreds individually. Other tissues are also envisioned to have utilityin the practice of the present invention. Explants can include matureembryos, immature embryos, meristems, callus tissue, or any other tissuethat is transformable and regenerable.

Any suitable plant culture medium can potentially be used during thetransformation process. Examples of such media would include but are notlimited to Murashige and Skoog (1962), N6 (Chu et al., 1975); Linsmaierand Skoog (1965); Uchimiya and Murashige (1962); Gamborg's media (1968),D medium (Duncan et al., 1985), McCown's Woody plant media (McCown andLloyd, 1981), Nitsch and Nitsch (1969), and Schenk and Hildebrandt(1972) or derivations of these media supplemented accordingly, as wellas the numerous media described below. Those of skill in the art areaware that media and media supplements such as nutrients and growthregulators for use in transformation and regeneration and other cultureconditions such as light intensity during incubation, pH, and incubationtemperatures can be optimized for the particular variety of interest.

Following regeneration of plantlets comprising shoots, or shoots androots, a selective agent may be applied to the plantlets, or parts ofplantlets, for instance if a selectable marker gene was beingtransformed into the initial target plant cells along with a GOI, or,alternatively, if the GOI itself encodes a selectable marker. Thus,after a plant has been produced by the methods of the present invention,a selective agent may be applied to it, in accordance with the presentinvention, in order to assist with assaying or otherwise identifying atransformed plant displaying useful characteristics.

The present invention also comprises methods for efficient handling ofregenerated plants, which allows identification of transformed plantscomprising the GOI. These methods simplify and streamline the process ofregenerating and growing the population of putatively transformedplants, saving time, space, and expense required by the process, andmaking the process commercially feasible.

In one embodiment, plant target tissue, such as immature corn embryos(IEs), is co-cultivated with an Agrobacterium strain comprising a GOI,for instance for 1-3 days at 23° C. followed by culturing at 30° C. foran additional ˜7-10 days on either the same co-culture medium or on acallus proliferation medium. Embryos may be observed in order toidentify which embryos are “responding” i.e. producing embryogeniccallus that may be regenerated to form a plantlet. Responding embryoswith callus, typically scutellar callus, are then transferred from theco-culture to a first pre-regeneration medium, or a first regenerationmedium, with appropriate culture conditions of temperature, light, andnutrients to allow further growth of callus, and differentiation andregeneration. The calli may be placed onto or into a semi-solidregeneration medium. Alternatively, they may be placed on a support,such as felt and/or filter paper in a culture plate, which is in contactwith a liquid regeneration medium, such that the callus can grow anddifferentiate.

As needed, the inoculated embryos may be cultured, for instance in thedark for 1-2 weeks at 30° C., including transfer to fresh nutrientmedium. After dark incubation, the cultures may be grown in regenerationmedia under alternating periods of light and darkness, for instance 1-3weeks of growth under a 16/8 light/dark cycle at about 27° C. with lightintensity of about 100 μE, or as appropriate based on the plant speciesor variety in question, and the knowledge of one of skill in the art ofplant tissue culture. Typically, initiation of plant regeneration beginswithin 1-3 weeks of the start of co-cultivation, especially if a callusphase of growth is present. The method may also comprise apre-regeneration step, which comprises use of a basal plant tissueculture medium supplemented with reduced levels of auxin(s) than is usedin callus proliferation medium.

After about 2-3 weeks of culture and regeneration on semi-solid orliquid media, the regenerating plants from a single explant, forinstance from immature embryos (IEs), may be transferred to a singlegrowth medium container. One such example is a PHYTATRAY (Sigma-Aldrich,St. Louis, Mo.) comprising either semi-solid or a liquid plant tissueculture regeneration medium and grown for about 4 weeks beforetransferring the resulting plants to growth media for hardening off theplants, such as growth plugs in soil. Because transplantation to soil isa time and labor intensive process, it may be preferable to screenindividual plants prior to their transplantation, or even prior to theirbeing placed in a PHYTATRAY, for the presence of the GOI or other traitof interest. The number of regenerated plantlets in the absence ofselective pressure may be 20-50× higher than would be found in a similarexperiment, using a selective agent during callus growth and plantregeneration. Thus, a process for handling the large number of plants,for instance for transferring them from the tissue culture phase togrowth under non-sterile conditions, is provided. Use of horticulturalplugs or individual culture tubes or trays under non-sterile conditionsto allow growth and analysis of plantlets is a further embodiment of theinvention. These plantlets may further be grown without necessarilylabeling all individual plants, by appropriate grouping of growingplants to allow easy correlation between a given plant and the itstissue which is being subjected to one or more assays or screens toidentify transformed plants comprising the GOI.

In one embodiment, a PCR-based screen may be employed to eliminatenon-transformed plants prior to their transfer to growth media,including liquid growth media, for instance in PHYTATRAYs. Thus, forexample, if about 5000 immature corn embryos are used in transformationwith a bacterial strain, about 25,000 plants may be produced, requiringabout 5,000 PHYTATRAYs. If about a 10% transformation frequency wasachieved, an initial screen of the regenerating plants at the PHYTATRAYgrowth stage would result in about 2500 putatively transformed plants,corresponding to 500 responding embryos or requiring about 500PHYTATRAYs, that would be transplanted to growth plugs in soil. Thescreening method may include pooling of tissues from regenerating plantsfrom individual Phytatrays or any other growth container such as plugs.The pools are designed such that, through analysis of multiple pools,single members of a population can be identified without the need forindividual analysis of each member of the population. One pooling methodis to group all plants derived from an explant, preferably an IE, in aPHYTATRAY or similar growth vessel and negative containers arediscarded, thereby greatly reducing efforts associated with planthandling and assaying. The number of plants pooled together could befurther increased, to the detection limit of a PCR assay.

The growth plugs may be handled or grouped to maximize the efficiency offurther screening steps, and to obviate the requirement for individuallylabeling the regenerated plants. For instance, the plugs may be groupedand oriented to correspond to an assay formatted to use a microtiterplate, for instance a 96-well plate by growing the plants in 96 pluggroups. This would allow rapid and accurate correlations to be madebetween the results of an assay and the plants from which assay tissuewas isolated. In certain embodiments, the assays to determine thepresence or absence of a GOI in a putatively transformed regeneratedmarker-free plant may be selected from the group consisting of aPCR-based assay, Southern hybridization, DNA sequencing, northernblotting, western blotting, an immunoassay, and an assay for anenzymatic activity encoded by the transgenic DNA segment which contactedthe target tissue during co-cultivation with Agrobacterium. In aparticular embodiment, the assay is a PCR-based assay. In certainembodiments, the PCR-based or other assay is performed on plant tissueisolated from regenerated plants growing in PHYTATRAYs or equivalent,prior to transplantation to a soil-based growth medium.

The present methods are more efficient than other typical methods forobtaining marker-free transgenic plants, for instanceAgrobacterium-mediated approaches using one or more T-DNA(s) comprisinga GOI, and a selectable or screenable marker (FIG. 1). Advantagesprovided by various embodiments of the invention include:

-   -   1. The transformation construct is smaller, simplifying the        cloning procedure.    -   2. Elimination of the marker gene expression cassette frees up        expression elements that would have been required for the marker        cassette, reducing concerns about recombinational stability due        to the presence of repeated elements. Elimination of repetitive        regulatory elements from the marker cassette also minimizes the        possibility of gene silencing.    -   3. The screening process for R₀ plants is simpler. In the        previous processes, at least two elements must be screened for,        the GOI and the selectable marker gene. In the present method,        there is no need to screen for a marker. Additionally, plants        positive for a GOI needed to be screened to determine whether        the marker gene insert is linked to the GOI insert, and often        linkage was found, which interferes with the ability to identify        plants lacking the selectable marker in a subsequent generation.        In contrast, linkage is not an issue in the present method.    -   4. Improved efficiencies in progeny generations is also found.        For prior methods such as 2-T transformation methods, a large        population of F₁ or R₁ plants must be screened to identify GOI        positive, marker-free plants. For plants produced by the present        method, no segregation of a marker gene is needed.    -   5. Allows for quicker selection of the best GOI-containing        events without the presence of the selectable marker gene        thereby facilitating efficient stacking of multiple GOI, e.g.        when the selectable marker gene encodes an agronomic trait of        interest.

The invention provides methods to efficiently produce marker-freetransgenic plants, generally capable of growth in a soil-based medium,within 7-10 weeks after an initial target explant is contacted by anexogenous nucleic acid. The high-throughput methods of the presentinvention allow development of an efficient transformation systemwithout selection. In particular, simplification of handling ofregenerating tissues and regenerated plants allows for mechanization ofmany steps, and saves time, money, and ergonomic burden. The system mayproduce about 4-6 usable marker-free transformation events (i.e. singlecopy events and vector backbone-free events) per transformationexperiment using about 100 embryos, thus expediting a transformed plantproduct pipeline.

EXAMPLES

The following examples are included to illustrate embodiments of theinvention. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the concept, spirit and scope ofthe invention. More specifically, it will be apparent that certainagents which are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Marker-Free Transformation

Transformation of regenerable immature corn embryos may be performed viaa Rhizobia-mediated protocol, e.g. as generally described by Cai et al.(U.S. Patent Application Publication 20040244075). In particular, amodified Agrobacterium-mediated method was used. Immature embryos with asize range of 1.9-2.5 mm, for instance about 2.3 mm, were selected fromcorn ears and co-cultivated with an ABI Agrobacterium strain C58 tomediate the transfer of DNA into the plant cells containing therecombinant construct of interest, for instance pMON93040 containingboth GUS and CP4 EPSPS under the expression control of an actinpromoter, to allow for both visual analysis of transformed cells andsectors, and to allow for use of a Weathermax™ glyphosate spray as asurrogate for a later, post-regeneration screen followed by aconfirmation test by a PCR-based screen for transformed plants. Largerembryos, e.g. about 2.5 mm or up to about 3.2 mm in size, may also beused, and may be preferable where few plants per embryo are produced byreducing callus proliferation before pre-regeneration, and regenerationphases of tissue culture. Composition of media used below are given inTable 1. Following inoculation with Agrobacterium, embryos weretransferred to Lynx 1947 or Lynx 1898 for co-culture for a period of 1-3days at 23° C., followed by additional 7-14 days at 30° C. on the sameplate or on a callus proliferation medium (e.g., Lynx 1316), followed bygrowing on a pre-regeneration medium (Lynx 1844; 2232; 2197) or aregeneration medium (Lynx 1344, 2282, 2379 etc. Final growth of theplants can be achieved by two methods: 1) transferring plants from eachembryo derived callus to a Phytatray™ containing Lynx 1607 or 2)transferring plants from each embryo derived callus to a Phytatray™containing liquid Lynx 2168. Plants were allowed to grow in Phytatray™for a period of about 4 wks before transferring them to plugs (Q Plugsby International Horticultural Technologies, Hollister, Calif.). Oneweek prior to transferring the plants to plugs, samples from the plantsare taken while the plants are still inside the Phytatray™ and assayedto remove plants without the GOI. Approximately, 10 days post-pluggingsamples from each plant were taken for DNA analysis and GOI positiveplants were identified and retained for further growth and development.

TABLE 1 Media compositions used in various aspects of the presentinvention. Function of representative media is identified. 1316 MediaComponents/L 1898 callus (Suppliers) co-culture 1947 proliferation 21332232 2197 2282 2379 MS Basal Salts 4.33 g 4.33 g 4.33 g 4.33 g 4.33 g4.33 g 4.33 g 4.33 g (Phytotech) MS Vitamins 10 mL 10 mL 10 mL 10 mL 10mL 10 mL 10 mL 10 mL (100X) (Phytotech) MSFromm 0 0 0 0 0 0 0 0 vitamins(1000X)* Thiamine HCL 0.5 mg 0.5 mg 0.5 mg 0.5 mg 0.5 mg 0.5 mg 0.5 mg 0(Sigma) 2,4-D 0.5 mg 0.5 mg 0.5 mg 0.5 mg 0.2 mg 0.2 mg 0 0 (Phytotech)Sucrose 30 g 30 g 30 g 30 g 50 g 50 g 50 g 60 g (Phytotech) Proline(Sigma) 1.38 g 1.38 g 1.38 g 1.38 g 1.38 g 0 0 0 Casamino Acids 0.5 g0.5 g 0.5 g 0.5 g 0.5 g 0.5 g 0.5 g 0 (Difco) pH   5.8   5.8   5.8   5.8  5.8   5.8   5.8   5.8 Low EEO 5.5 g 5.5 g 0 5.5 g 5.5 g 0 0 0 Agarose(Sigma) Phytagel (Sigma) 0 0 3.0 g 0 0 0 0 0 Phytagar (Gibco) 0 0 0 0 00 0 0 Post autoclave additives Carbenicillin 50 mg 50 mg 500 mg 500 mg50 mg 500 mg 500 mg 500 mg (Phytotech) Acetosyringone 200 uM 200 uM 0200 uM 200 uM 0 0 0 (Aldrich) BAP (Sigma) 0 0.01 mg 0.01 mg 0.01 mg 0.01mg 0.01 mg 0.01 mg 0.01 mg Glyphosate 0 0 0 0 0 0 0 0 (Gateway Chemical)Silver Nitrate 3.4 mg 3.4 mg 3.4 mg 3.4 mg 3.4 mg 3.4 mg 3.4 mg 0(Sigma) Abscisic acid 0 0 0 0 0 0 0 0 Media Components/L 1844 1344 1607(Suppliers) pre-regeneration regeneration 2168 growth 1471 MS BasalSalts 4.33 g 4.33 g 4.33 g 4.33 g 4.33 g (Phytotech) MS Vitamins 10 mL10 mL 10 mL 10 mL 10 mL (100X) (Phytotech) MSFromm 0 1 mL 0 0 0 vitamins(1000X)* Thiamine HCL 0 0 0 0 0 (Sigma) 2,4-D 0.2 mg 0 0 0 0 (Phytotech)Sucrose 40 g 30 g 60 g 60 g 60 g (Phytotech) Proline (Sigma) 0 1.38 g 00 0 Casamino Acids 0 0.5 g 0 0 0 (Difco) pH   5.8   5.8   5.8   5.8  5.8 Low EEO 0 0 0 0 0 Agarose (Sigma) Phytagel (Sigma) 0 3.0 g 0 0 0Phytagar (Gibco) 6 0 0 6 6 Carbenicillin 500 mg 250 mg 500 mg 100 mg 100mg (Phytotech) Acetosyringone 0 0 0 0 0 (Aldrich) BAP (Sigma) 0 3.5 mg 00 0.02 mM Glyphosate 0 0 0 0 0 (Gateway Chemical) Silver Nitrate 0 0 0 00 (Sigma) Abscisic acid 0.26 mg 0 0 0 0 *1000X stock contains Nicotinicacid −1.25 g; Pyridoxine HCL 0.25 g; Thiamine HCl 0.25 g; CalciumPantothenate 0.25 g

Example 2 Efficient Development of Transgenic Sectors without SelectionPressure During Callus Proliferation

A system for efficient regeneration of transgenic plants in the absenceof a selection agent was developed. Following co-culture of an explantwith Agrobacterium (4 days on Lynx 1898 medium (Table 1), callusproliferation commenced on Lynx 1316 (Table 1), for 10-14 days, withoutselection. Next, pre-regeneration of callus tissue was performed for 10days on Lynx 1844 medium (Table 1), followed by regeneration on Lynx1344 (Table 1) for 10 days, and Lynx 1471 for 3 weeks (Table 1). Allsteps except for culture on Lynx 1471 were performed without use of aselective agent; thus callus growth and plant regeneration occurredwithout a selective agent for about 4 weeks after co-cultivation. Growthof regenerating plants in the last step, on Lynx 1471, was performed inthe presence of a low level of glyphosate (0.02 mM, v/v) to estimate themaximum possible transformation frequency. Prior to transferring tissuesto Lynx 1471 media, 24 independent embryo-derived calli and associatedtissues were stained for GUS activity at 4 weeks post-transformation.Four GUS positive shoots were identified, thus demonstrating ˜16%transformation efficiency.

Further plant growth was achieved by transferring tissues to Lynx 1471in Phytatrays, and a total of 43 transgenic events were regenerated, allof which survived upon transfer to soil. The transformation and copynumber analysis is shown in Table 2. About 14% of the total plants thatsurvived were escapes, but about 45% of the plants were transformed with1-2 inserts. Histochemical analysis of representative regeneratingcallus lines, 5 weeks post-transformation is shown in FIG. 2.

TABLE 2 Efficient transformation using selection only during last stepof plant regeneration indicates efficient transformed sector formationwithout selection. # # to soil 1 copy and 2 copy and Expt Explants(survived) 0-copy 1-copy 2-copy >2copy oriV minus oriV minus 6678 200 43(21.5%) 6 (14%) 12 (28%) 7 (16%) 8 (19%) 9 (21%) 4 (9%)

Example 3 Additional Corn Transformation and Regeneration Experiments,and Screening of Putative Transformed Plants

Three more studies were performed to confirm that efficient regenerationof transgenic sectors was routinely possible without applying selectionat any stage. The plasmid used was pMON93040, described above. Followingco-culture on Lynx 1898 for 1 day, callus proliferation was performed onLynx 1316 for 10 days, pre-regeneration on Lynx 1844 for 10 days, andregeneration on Lynx 1344 for 3 days, followed by growth on Lynx 1607. Asingle corn ear was used to isolate the embryos for each experiment, andthe embryos ranged in size from 2.8-3.2 mm. In two of the studies,embryo inoculation was performed directly isolating embryos intoAgrobacterium suspension at O.D.₆₆₀=1.0, while in the other studyembryos were first isolated into 1 ml of liquid Lynx 1013 medium (1Liter: MS Basal Salts (Phytotech): 2.165 g; MS Vitamins (100×;Phytotech): 10 ml; Sucrose (Phytotech): 68.5 g; Proline (Fisher): 0.115g.; Glucose (Phytotech) 36 g. The medium was adjusted to pH 5.4 withKOH, and filter sterilized), followed by inoculation using anAgrobacterium suspension at O.D.₆₆₀=1.0. Results of the studies arelisted in Table 3.

TABLE 3 Additional Corn Transformation without Selection. # plants in #gus/CP4 #Events/100 plants Expt # IES plugs positive plants (estimated)6688-2 110 (48)* 230 8 3.5 6698-2 110 (52)* 240 9 3.8 6700-2 100 (50)*253 20 7.9 Average/100 plants 5.1 *data in parenthesis indicates # ofresponding embryos; ~50% embryos responded to culture “# IES” = numberof inoculated immature embryos

At the end of the regeneration cycle, plants from each experiment weretransplanted into plugs and over 95% plants survived the transfer,demonstrating that propagation plugs offer an improved way to handlelarge number of plants. About ten days post transplanting, leaf punchesfrom individual plants were assayed for GUS activity using histochemicalstaining, and GUS positive plants were transplanted for further growthand histochemical analysis. To further demonstrate the transformationfrequency and to improve the overall efficiency of the protocol, asurrogate for a PCR-based screen was developed, whereby 1% WeatherMax™(Glyphosate) was applied to GUS negative plants. An additional fiveglyphosate tolerant plants were identified (3 from experiment 6700-2, 1from 6698-2 and 1 from experiment 6688-2 (see FIG. 3 for representativeplants). A total of 37 plants were obtained from 723 plants in plugs,yielding an estimated 5% success rate on a per plant basis (Table 3).The results of transformation without selection, using immature embryosdemonstrate that the process is efficient, with an average 5%transformation frequency based on the number of plants screened.

To further demonstrate reproducibility of the transformation protocolwithout selection and to improve the overall efficiency of the protocol,a surrogate for a PCR-based screen was developed, whereby 1% WeatherMax™(Glyphosate) was applied after the end of the regeneration phase whichwas carried out without selection. in PHYTATRAYs. Results are shown inTable 4 and Table 5.

TABLE 4 Efficient and Reproducible Corn Transformation without Selection# #Events/ embryos # Esti- 100 plants inocu- IE phytas mated # #cp4+(esti- Expt lated size screened plants events mated) 6705-1 130 2.8-3.219 380 9 2.4 6705-2 140 2.8-3.2 13 260 7 2.7 6706-3 110 1.8-2.0 22 44035 8 6829-1 100 2.8-3.0 28 560 27 4.8 6829-2 100 2.8-3.0 30 600 43 7.26829-3 80 2.8-3.2 10 200 11 5.5 6829-4 80 2.8-3.2 9 180 3 1.7 6829-5 802.8-3.2 8 160 4 2.5 6829-6 80 2.8-3.2 7 140 2 1.4 900 146 2920 141 4.8

TABLE 5 Efficient Recovery of Events with Lower Copy Inserts fromTransformation without Selection. # embryos # Events Total Exptinoculated survived in plugs (1-2 copy) 6705-1 130 9 7 (77.7%) 6705-2140 7 2 (28.6%) 6706-3 110 35 29 (82.9%)  6829-1 100 27 18 (66.7%) 6829-2 100 43 30 (69.8%)  6829-3 80 11 8 (72.2%) 6829-4 80 3 2 (66.7%)6829-5 80 4 3 (75.0%) 6829-6 80 2 1 (50.0%) Total 900 141 100 (70.9%) 

The results indicate that out of a total of 900 embryos, 141 glyphosatetolerant plants were produced, including 100 with lower copy number (1-2copies) of the gene of interest, i.e. an average of about 15% TF basedon the number of immature embryos inoculated.

Further screening (Table 6) showed that, of the 90 events screened bySouthern analysis out of the 100 low copy number events shown in Table5, 79 independent integration events were present. Sister events areevents with same band pattern and coming from the same explants. Highertransformation frequency lead to higher percentage of transgenic eventswith sister events. Nevertheless, the frequency was very low andSouthern analysis revealed ˜5% exhibiting clonality, especially when TFis >30% (Tables 5 and 6).

TABLE 6 Efficiency of Transformation without Selection- Number ofIndependent Integration Events Produced. # Events analyzed by # Eventswith Expt Southern blot (1 & 2 copy) sister 6705-1 1 0 6705-2 7 0 6706-325 3 6829-1 16 1 6829-2 27 1 6829-3 8 0 6829-4 2 0 6829-5 3 0 6829-6 1 0Total 90 5

Example 4 Confirmation of Chromosome Integration of a Transferred DNAFollowing Transformation and Regeneration Without Selective Pressure

Genomic DNA was isolated from leaves of R₀ plants, e.g. using proceduresdescribed by Dellaporta (1983). Genomic DNA (20-30 μg) was digested withHindIII, separated on a 0.7% (w/v) agarose gel, and transferred topositively charged nylon membranes (Roche Molecular Biochemicals,Indianapolis, Ind.). Pre-hybridization, hybridization, washing anddetection of the membranes were conducted using a non-radioactiveDIG-based system (Roche Molecular Biochemicals) following themanufacturer's protocols. DNA sequence from the CP4 gene was labeled byPCR to produce probes. The HindIII enzyme cuts once with in the vector(near the 5′ end of the CP4 expression cassette, therefore, the numberof bands by Southern blot corresponds to the number of CP4 gene copies.Southern analysis was performed on selected plants described in Table 3(i.e. 22 lower copy events were selected). Representative Southernanalysis data of R₀ plants is shown in FIG. 4. Analysis revealed thateight (single copy, oriV negative) events were produced from thepopulation of 37 events produced from 320 embryos (Table 3). To furtherconfirm germline transmission to the next generation, R₀ plants werecrossed with the parental non-transformed inbred corn line. In thisstudy, R₁ plants from three independent lines were used; ZM_(—)187694 (4copies-cp4); ZM_(—)189983 (0 copy, cp-4—a possible cp4 truncated event);ZM_(—)187738 (3 copies—cp4). Histochemical analysis of gus expression ofthe developing ears indicated that positive kernels to non-expressingkernels in 1:1 ratio, indicating germline transmission of the transgeneand linkage (FIG. 5). This result confirms germline transmission of atransgene using transgenic events produced without selection. Progenyfrom three additional independent events ZM_(—)187692, ZM_(—)18997, andZM_(—)18998 were also analyzed by Southern blot analysis using CP4 geneas a probe (FIG. 6), showing that all progenies derived from threedifferent R₀ plants showed the expected pattern, i.e. stabletransmission of the transgenic event to the next generation.

Example 5 Efficient Plant Handling

A method for efficient handling of multiple plants is an importantcomponent of an efficient plant transformation system that does not usea selective agent prior to obtaining a regenerated plant. This isbecause a large number of plants may need to be screened in order toidentify transformed plants with appropriate copy number and complexityof insertions, as well as expression of the GOI. This “handling” (e.g.transfer or transplantation to media or soil for further growth; andmaintenance of identity during screening steps) allows multipleplantlets to be processed inside a container holding the individualplugs or culture tubes, while maintaining the plants' individualindentity, and also facilitates data capture without labelling ofindividual plants. The combination of growing plants in “horticulturalplugs,” skipping labelling of individual plants, and developing aprotocol to capture assay data for identifying and moving forward thedesired events, expedites the transformation pipeline based onnon-selection during gene transfer and regeneration. In summary thepresent invention relates to the development of an efficient planttransformation system without selection, plant handling and datacapture.

Putatively transformed plants, regenerated from calli that have beenco-cultivated with an Agrobacterium strain comprising a gene ofinterest, can be transplanted, e.g. from a Phytatray™ into soil ingrowth plugs. Use of these plugs can streamline sampling and analysis ofthe plants, and save growth space. For instance, the plugs are arrangedin a pattern that corresponds to the wells of, for instance, a 96 wellmicrotiter plate (e.g. FIG. 7), if assays of samples from the plants areto be performed in such microtiter plates. This allows facileidentification of plants displaying an assay phenotype of interestwithout the need to label individual plants.

Early elimination of plants not comprising the GOI is accomplished by aPCR-based or other molecular screen while plants are being regeneratedin PHYTATRAYs on semi-solid or liquid media, and prior totransplantation of plants to growth plugs or soil.

Example 6 Semi-Solid Media for Culture During Regeneration ofTransformed Plants

Semi-solid media for culture during the callus growth, pre-regeneration,and regeneration phases of the transformation and tissue culture processallows efficient tissue manipulation. FIG. 8 summarizes a transformationstudy carried out without a selective agent (lower panel), as comparedto a parallel study wherein plant tissue was grown in the presence of aselective agent (top panel). See Tables 1 and 7 for media components.Callus proliferation, pre-regeneration, and regeneration phases werecarried out in semi-solid media as shown. After the second regenerationphase, plants are transplanted into growth plugs and assayed for thepresence of a GOI.

TABLE 7 Media compositions used in a previous method comprisingsemi-solid glyphosate containing selection media (Cai et al.; U.S.patent application Pub. No. 2004/00244075). 1073 Media Components/L 12331278 (1^(st) 1071 1084 (Suppliers) (co-culture) (selection) regeneration(2^(nd) regeneration) (rooting) MS Basal Salts (Phytotech) 2.165 g 4.33g 4.33 g 4.33 g 2.165 g MS Vitamins (100X) 10 mL 10 mL 0 0 0 (Phytotech)MS Fromm Vitamins 0 0 1 mL 1 mL 0 (1000X)* BAP (Sigma) 0 0.01 mg 3.5 mg0 0 Thiamine HCL (Sigma) 0.5 mg 0.5 mg 0 0 0 2,4-D (Phytotech) 3 mg 0.5mg 0 0 0 NAA (Sigma) 0 0 0 0 0.5 mg IBA (Sigma) 0 0 0 0 0.75 mg Sucrose(Phytotech) 20 g 30 g 30 g 0 20 g Glucose (Phytotech) 10 g 0 0 10 g 0Maltose (Phytotech) 0 0 0 20 g 0 Proline (Sigma) 115 mg 1.38 g 1.38 g 00 Casamino Acids (Difco) 0 0.5 g 0.05 g   0.5 0 Asparagine monohydrate 00 0   0.15 0 (Sigma) Myo-inositol (Sigma) 0 0 0 0.1 g 0 Low EEO Agarose(Sigma) 5.5 g 0 0 0 0 Phytagel (Sigma) 0 3 g 3 g 3 g 3 g Acetosyringone(Aldrich) 200 uM 0 0 0 0 Carbenicillin (Phytotech) 500 mg 500 mg 250 mg250 mg 0 Glyphosate (Gateway 0 0.1 mM 0.1 mM 0.1 mM 0.1 mM Chemical)Silver Nitrate (Sigma) 3.4 mg 3.4 mg 0 0 0 pH   5.2   5.8   5.8   5.8  5.8 *Comprising 1250 mg/L nicotinic acid (Sigma), 250 mg/L pyridoxineHCl (Sigma), 250 mg/L thiamine HCl (Sigma), and 250 mg/L calciumpantothenate (Sigma).

Example 7 Liquid Culture During Regeneration of Transformed Plants

Liquid culture during the callus growth, pre-regeneration, andregeneration phases of the transformation and tissue culture processenables efficient tissue manipulation. FIG. 9 illustrates transformationand regeneration studies carried out without a selective agent prior toregeneration. Callus proliferation, pre-regeneration, and regenerationphases were carried out in liquid, glyphosate-free media as shown. Afterthe second regeneration phase, which may alternatively occur in asemisolid medium, plants were transplanted into growth plugs. GUShistochemical assays may be combined with PCR-based or other screens,such as a surrogate screen with, for instance, glyphosate, to detectexpression of, for instance, glyphosate tolerance in the regeneratedplant tissue. These experiments were performed either using a vectorwithout a marker gene (i.e. pMON97372) or with a marker gene(pMON93040).

The plasmid for the study illustrated in FIG. 9 was pMON93040 containingcp4 and gus genes. Embryos from each ear were isolated into Petri disheswith 1 ml of liquid Lynx 1013 medium and co-cultured on Lynx 1947.Embryos were divided among various treatments as shown in the Table 8below, including 8 weeks with selection (Treatment 1); 8 weeks withoutselection, liquid culture, with growth in solid medium in PHYTATRAY(Treatment 2); and 8 weeks without selection, liquid culture, withgrowth in liquid medium in PHYTATRAY (Treatment 3). Transformation usingno selection was quite efficient (˜⅓×) compared to what was achievedusing selection. A summary of the results of the “liquid-plug” methodare shown in Table 8. When a small sample of explants was carriedforward using only liquid culture (i.e. treatment #3, using Lynx 2168 asthe final growth medium), the efficiency was higher and nearly as highas what was obtained with selection. Liquid culture appears to promotemore efficient regeneration of transgenic events as evident from Table9. It is likely that elimination of sub-culture reduces stress andhastens plant regeneration.

TABLE 8 Efficient transformation using “liquid-plug scheme” and withoutusing selection. # Explants # IE with Exp# Treatment IE Size (IEs)events % TF 7530-1 Liquid Selection - 2.5-2.8 30 14 46.7 7531-1 8 wks1.9-2.1 30 16 53.3 7532-1 1.9-2.2 30 4 13.3 7533-1 2.5-2.8 30 7 23.3Total 120 41 34.2 7529-1 Transformation 2 60 5 8.3 7530-2 withoutselection using 2.5-2.8 88 16 18.2 7531-2 liquid culture with a 1.9-2.168 13 19.1 7532-2 PHYTATRAY step + 1.9-2.2 127 11 8.7 glyphosate sprayscreening Total 343 45 13.1 7530-3 Transformation 2.5-2.8 21 9 42.97531-3 without selection using 1.9-2.1 18 10 55.6 7532-3 liquid culturewith 1.9-2.2 22 4 18.2 glyphosate spray screening after transplanting inplugs Total 61 23 37.7

TABLE 9 Efficiency of transformation as related to regenerationefficiency. # # IE # Explants with Plants #Events/ Exp# Treatment (IEs)events % TF in plugs 100 plants 7530-3 Transforma- 21 9 42.9 208 4.37531-3 tion without 18 10 55.6 208 4.8 7532-3 selection 22 4 18.2 2081.9 using liquid culture with glyphosate spray screening after trans-planting in plugs Total 61 23 37.7 624 3.7

Example 8 Transformation Efficiency as Related to Duration of CallusProliferation Phase

The role of the duration of the callus proliferation phase ontransformation frequency is illustrated in Table 10. Reducing the lengthof the callus phase improved TF, producing plants faster.

TABLE 10 Longer callus proliferation phase negatively impactstransformation without selection. IE # To # IE with Exp# Treatment Sizesel events % TF 7533-1 Sel - 8 wks 2.5-2.8 30 7 23.3 liquid culture.7533-2 No selection - 2.5-2.8 104 8 7.7 8 wks (2 wks on Lynx 2133)7533-3 No selection - 2.5-2.8 97 19 19.6 8 wks (1 wk on Lynx 2133)

Example 9 Transformation and Regeneration Using Rapid Liquid Culturewithout Selection

To further streamline the process, a rapid liquid cycle (RLC) protocolwas developed, 6 weeks in length, wherein the callus proliferation step(Lynx 2133) was omitted. The steps in the transformation schemeincluded: pre-regeneration (Lynx 2197), and regeneration phases (Lynx2168 and Lynx 1607 e.g. FIG. 9 and Table 11). pMON93040, containing Cp4and gus genes, was used for this study. Embryos from each ear wereisolated onto a Petri dish with liquid medium (Lynx 1013), co-culturedon Lynx 1947 medium, and divided among the treatments shown in Table 11.Treatments included comparing TF of embryos with or without selection,and comparing the effect of either 1 or 2 weeks on Lynx 2197proliferation medium. As shown in Table 11, a reduction of duration onLynx 2197 proliferation medium did affect TF. However, transformationwithout selection resulted in TF comparable to the TF achieved usingselection when an optimum duration of growth on pre-regeneration mediumwas used.

TABLE 11 Efficient Transformation Using Rapid Liquid Culture Protocol,without Selection. # # # IE plants Events/ Explants with % to 100 Exp#Treatment (IEs) events TF plugs plants 7962-3 Sel - 6 wks liq. 65 3756.9 7963-1 65 16 24.6 N/A N/A Total 8 wks liq sel 130 53 40.8 7965-1 6wks No selec- 60 27 45.0 548 4.9 7963-2 tion with 2 wks 60 18 30.0 342 7on Lynx 2197 Total No Sel. 2 wks 120 45 37.5 890 5.7 Lynx 2197 7965-1 6wks No selec- 60 12 20 493 2.4 7963-2 tion with 1 wk 60 13 21.7 274 4.7on Lynx 2197 Total No Sel. 1 wk 120 25 20.8 767 3.2 Lynx 2197

In these experiments, the duration of the callus proliferation,pre-regeneration, and regeneration phases were further reduced, and useof selection was compared to growth on non-selective media. Thereduction in required tissue handling steps further renders this methodamenable to automation (See also Table 15).

A comparison of the expression level of selected plants obtained asdescribed above revealed comparable levels of expression (Table 12)following PCR-based assay for the pinII 3′ transcription terminationsignal.

TABLE 12 Expression analysis of plants categorized by copy number oftransgene. Average Expression Copy # for PinII # Plants value for PinIITreatment 3′ UTR assayed 3′ UTR SD RLC with 1 70 1.432 1.5 selection 228 1.902 2.6 RLC with no 1 44 0.957 3.37 selection* 2 35 0.809 1.3*Plants from “no selection” were 2 weeks older at the time of sampling

A summary timeline of the rapid liquid culture protocol withoutselection is given in Table 13.

TABLE 13 Rapid liquid culture protocol without selection. Days Medium #Culture conditions 0 d Lynx 1947 1 day @23° C., dark; 6 days at 30° C.,dark 1^(st) week Lynx 2197/2379/2282 30° C., dark 2^(nd) week Lynx 216830° C., dark 3^(rd) week Lynx 2168 27-28° C., light 4^(th) week Lynx2168 27-28° C., light 5^(th) week Lynx 1607 Transfer explants toPHYTATRAYs ~8^(th) week Transplant Harden off plants 10 days post Samplefor glyphosate tolerance or transplanting for GUS assay or for PCT assayfor GOI 20 days post Advance Assign event numbers and transplantingadvance the positive events

To validate the above results based on the protocol described in Table13, three experiments were conducted using transformation withpMON93040. Embryos were divided among various treatments as shown in theTable 14. In these experiments GUS assays were performed on plantlets;glyphosate was not sprayed. Embryos from each ear were isolated into aPetri-dish with 1 ml of liquid Lynx 1013 medium and co-cultured on Lynx1947. As evident from the results, transformation using no selection isquite efficient (>60%) compared to what was achieved using selection.

TABLE 14 RAPID Transformation protocol works efficiently with bothselection and no selection. # explants # # Events/ Ave Expt # Treatment(IE's) Events TF (%) Plants 100 plants SD plants/IE 8105-4 RLC, 55 2443.6 N/A 8106-4 Selection 80 34 42.5 8107-4 60 27 45 Average 195 85 43.68108-1 RLC, No 75 23 30.7 347 6.6 3.3 5.5 8108-2 selection 60 18 30 3874.7 3.5 6.5 8108-3 60 14 23.3 373 3.8 3.3 7.2 Average 195 55 28.2 369 53.4 6.4

In order to better understand the nature of cell proliferation and it'seffect on transformation without selection, additional experiments wereconducted using two different co-culture media (Lynx 1947 with 0.5 mg/l2,4-D and Lynx 2232 (with 0.2 mg/l 2,4-D). Embryos from each co-culturemedium were equally divided into two groups and either transferred topre-regeneration medium (with 0.2 mg/l 2,4-D—Lynx 2197) or regenerationmedium (without any growth regulators—Lynx 2282). Five experiments(8415-8419) were conducted using a marker-less transformation vector(pMON97372) containing only the uidA gene. An outline of theexperimental approach is given in FIG. 9. After establishing plants inplugs, plants from each of the embryo-derived lines were pooled andstained for GUS to identify positive lines. Later, further GUS stainingof individual lines from the positive clones was performed to identifyGUS positive events. About ⅕^(th) embryos from each ear were separatelyinoculated with a control uidA+cp4 vector (pMON97367) and the RLCprotocol (e.g. Table 13) was followed to compare transformation resultswith and without selection (experiment 8420). The results from theseexperiments are summarized in Tables 15 and 16.

TABLE 15 Efficient transformation without selection using a gus vector(pMON 97372). # Total # # IE % TF % TF IE Explants # To plants to with(based on # (based on # Expt # Treatment size (IEs) Phytatrays PlugsEvents to selection) Phytatrays) 8415-1 Co-culture on 2.3 60 30 273 9 1530 8416-1 Lynx 1947 - 1st 2 60 37 273 9 15 24 8417-1 regeneration 2.4 6042 281 3 5 7.1 8418-1 on Lynx 2197 1.9 60 40 342 8 13.3 20 8419-1 2.1 6037 405 7 11.7 18.9 Total 300 186 1574 36 12 19.4 8415-2 Co-culture on2.3 60 34 295 9 15 26.5 8416-2 Lynx 1947 - 1st 2 60 34 274 7 11.7 20.68417-2 regeneration 2.4 60 45 290 7 11.7 15.6 8418-2 on Lynx2282 1.9 6034 232 9 15 26.5 8419-2 2.1 60 41 445 5 8.3 12.2 Total 300 188 1574 3712.3 19.7 8416-3 Co-culture on 2 45 36 398 7 15.6 19.4 8417-3 Lynx2232 - 1st 2.4 45 28 239 3 6.7 10.7 8418-3 regeneration 1.9 45 22 276 511.1 22.7 8419-3 on Lynx 2197 2.1 40 37 256 4 10 10.8 Total 175 123 116937 10.9 15.4 8416-4 Co-culture on 2 45 28 297 2 4.4 7.1 8417-4 Lynx2232 - 1st 2.4 45 30 208 4 8.9 13.3 8418-4 regeneration 1.9 45 33 226 48.9 12.1 8419-4 on Lynx 2282 2.1 45 35 342 6 13.3 17.1 Total 180 1261073 16 8.9 12.7

TABLE 16 Transformation experiments using a (pMON97367) with selectionindicates transformation without selection is efficient. IE # IE % TFControl size # to with (based on Expt # Treatment for (mm) sel Events #to sel) 8420-1 Transformation 8415 2.3 50 6 12 8420-2 using RLC with8416 2 40 5 12.5 8420-3 pMon97367 8417 2.4 40 4 10 8420-4 8418 1.9 50 48 8420-5 8419 2.1 50 3 6 Total 230 22 9.6

As evident from the Table 15, co-culture medium Lynx 1947, with higher2,4-D concentration than Lynx 2232, was found to be superior. Theseresults indicate that cell proliferation prior to regenerationcontributes to obtaining efficient transformation without selection.Furthermore, regeneration of explants on a growth-regulator free medium(e.g. Lynx 2282) following co-culture did not appreciably reduce thenumber of plants/embryo. Comparison of transformation frequency withembryos that were transformed with pMON97367 using the RLC protocoldemonstrates transformation without selection to be efficient.Furthermore, copy number analysis indicated a higher percentage ofplants with lower copy insert (Table 17).

TABLE 17 Transformation without selection resulted in higher % of singlecopy, oriV negative events. Total # of Plants Assayed % Single CopyEvents, lacking oriV 122 66 (55%)

It is thought that the absence of selection, and/or the shorter T-DNAmight be reasons for the higher % usable event production. For the noselection treatments, the preferred embryo size range is slightly larger(2.0-2.3 mm) than the embryo size range (1.9-2.1 mm) used for RLC.Validation by Southern hybridization of ˜120 events (Table 17) indicatedthat all events are independent. This further emphasizes the earlierfinding that under low TF (˜15%) most of the events produced areindependent events indicating that adoption of a pooling strategy priorto transplanting to growth plugs can further improve the efficiency ofthe protocol.

Example 10 Screening Plants Before Transplanting to Plugs to EliminateNon-Transgenic Plants Improves Efficiency

Plants from a single container containing the final growth medium (Lynx1607) were pooled together and assayed for both gus using thehistochemical GUS assay and the presence of the transgene using PCR. A100% correlation between GUS and PCR assays was obtained. This studyallowed the elimination of growth containers without positive event,thereby greatly reducing plant handling burden and improvingthrough-put.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The references listed below are incorporated herein by reference to theextent that they supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

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What is claimed is:
 1. A method for identifying transgenic corn plants,comprising: (a) obtaining corn plant cells transformed with a DNAsegment comprising a nucleic acid sequence of interest; (b) regeneratinga plurality of corn plants or differentiated corn plant parts from thecells without first selecting for the presence of said DNA segment; (c)identifying at least a first transgenic corn plant or transgenicdifferentiated plant part from the plurality of corn plants ordifferentiated corn plant parts.
 2. The method of claim 1, wherein theDNA segment does not comprise a selectable marker or visual marker gene.3. The method of claim 1, wherein the plants are regenerated by growthon solid media, liquid media, or a combination of solid and liquidmedia.
 4. The method of claim 3, wherein the plants are regenerated bygrowth solely on liquid media prior to identifying the transgenic cornplant or transgenic differentiated plant part.
 5. The method of claim 4,wherein the transformation frequency of cells grown solely in liquidmedia subsequent to contacting the cells with a GOI and prior toidentification of transgenic plants or transgenic plant parts isenhanced relative to the transformation frequency observed when cellsare grown in solid media, semi-solid media, soil, or a combination ofsolid media, semi-solid media, liquid media, and/or soil, subsequent tocontacting the cells with a GOI and prior to identification oftransgenic plants or transgenic plant parts.
 6. The method of claim 1,wherein the plant cells are immature corn embryo cells.
 7. The method ofclaim 6, wherein the immature corn embryos are from about 1.5 mm toabout 3.5 mm in length.
 8. The method of claim 7, wherein the immaturecorn embryos are from about 1.9 mm to about 2.3 mm in length.
 9. Themethod of claim 1, further comprising, between steps (b) and (c): (1)placing the plurality of corn plants or differentiated plant parts inculture tubes or growth plugs comprising a growth medium or water whilemaintaining the individual identity of the corn plants; and (2)subjecting the plants or plant parts to at least a first assay for thepresence of the DNA segment to identify one or more plant or plant partas transgenic based on results from the assay.
 10. The method of claim9, wherein the assay is selected from the group consisting of Southernhybridization, PCR, DNA sequencing, northern blotting, western blotting,an immunoassay, and an assay for the enzymatic activity encoded by theDNA segment.
 11. The method of claim 9, wherein the assay is performedprior to placing the regenerated plants into soil.
 12. The method ofclaim 10, wherein putatively transformed corn plants or differentiatedplant parts lacking the nucleic acid sequence of interest areidentified, wherein the assay is performed on plant tissue comprisingpooled subsets of nucleic acids from said plurality of corn plants ordifferentiated plant parts.
 13. The method of claim 1, wherein the cornplants or corn plant parts are regenerated not later than 6 weeks afterthe DNA segment is transformed into the corn plant cells.
 14. The methodof claim 1, wherein the corn plants or corn plant parts are regeneratednot later than 4 weeks after the DNA segment is transformed into thecorn plant cells.
 15. The method of claim 1, wherein the corn plants orcorn plant parts are regenerated not later than 3 weeks after the DNAsegment is transformed into the corn plant cells.
 16. The method ofclaim 1, wherein the corn plants or corn plant parts are regenerated notlater than 2 weeks after the DNA segment is transformed into the cornplant cells.
 17. The method of claim 1, wherein the corn plants or cornplant parts are regenerated not later than 1 week after the DNA segmentis transformed into the corn plant cells
 18. The method of claim 1,wherein the DNA segment is introduced into the corn plant cell bybacterially-mediated transformation, electroporation, PEG-mediatedtransformation, or particle bombardment.
 19. The method of claim 18,wherein the bacterially-mediated transformation is mediated by abacterial cell selected from the group consisting of an Agrobacteriumcell, a Rhizobium cell, a Sinorhizobium cell, and a Mesorhizobium cell.20. The method of claim 1, further comprising the step of subjecting acorn plant or plant part derived from the first corn plant cell toculture conditions that select for, or allow screening for, the presenceor absence of the nucleic acid sequence of interest after regenerationof a plant or plant part.
 21. The method of claim 1, wherein the growthmedium is a solid medium.
 22. The method of claim 21, wherein the growthmedium is soil.
 23. The method of claim 1, wherein the regenerated plantor differentiated plant part is uniform with respect to the presence ofthe DNA segment